The present invention relates to a method for collecting DNA contained in microorganisms.
In the genetic engineering, plasmid DNA is isolated from microorganisms by transforming microorganisms such as E. coli, culturing the transformants and collecting des red plasmid DNA from the amplified transformants.
However, collection and purification of plasmid DNA from transformants require several steps and are tedious. Many improvements have been proposed in methods for purification of plasmid DNA.
For example, Japanese Patent Unexamined Publication No. Hei 4-360686 (JP-A-360686/92) discloses a method for the purification of plasmid DNA and/or cosmid DNA by lysing microbial cells, filtering the resulting lysate with a membrane filter to remove insolubles and subjecting the filtrate to ultrafiltration to remove impurities and concentrate the DNA.
Japanese Patent Unexamined Publication No. Hei 8-23976 (JP-A-23976/96) discloses a method for purifying supercoiled plasmid by removing impurities from a plasmid mixture using a filtration filter having an average pore diameter of 10 nm to 35 nm.
In these methods, however, the resulting purified DNA may contain RNA, which has been contained in the microbial cells together with the DNA, and an additional step is required to degrade the RNA to provide DNA without contamination.
There has been known a method for separating RNA and DNA utilizing a carrier capable of adsorbing DNA together with a chaotropic solution (Chaotropic Ion Method) (R. Room et al,. J. Clin. MicroBiol. Vol.28, No.3, p495-503). Japanese Patent Unexamined Publication No. Hei 7-250681 (JP-A-250681/95) discloses a method for purifying DNA in which RNA contained in microbial cells with the DNA is removed by the above method.
This method is one for extracting and purifying plasmid DNA comprising steps of collecting microbial cells from a culture of transformants into a first cartridge, lysing the cells and separating undesired RNA, filtering off impurities by the first cartridge, and adsorbing, washing and eluting the DNA by a second cartridge.
However, this method requires two cartridges, and the first cartridge should have at least a trap filter and a membrane filter, and the second should have at least a glass fiber filter, glass powder layer and membrane filter. These cartridges are structurally more complex compared to a simple filter itself. In addition, this method requires repetitive feeding and draining of the solution by aspiration using the two cartridges.
Therefore, an object of the present invention is to provide a method for collecting DNA by the Chaotropic Ion Method with an apparatus with simpler structure and fewer operations.